A Comparison of Assays for Accurate Copy Number Measurement of the Low-Affinity Fc Gamma Receptor Genes FCGR3A and FCGR3B

Abstract

The FCGR3 locus encoding the low affinity activating receptor FcγRIII, plays a vital role in immunity triggered by cellular effector and regulatory functions. Copy number of the genes FCGR3A and FCGR3B has previously been reported to affect susceptibility to several autoimmune diseases and chronic inflammatory conditions. However, such genetic association studies often yield inconsistent results; hence require assays that are robust with low error rate. We investigated the accuracy and efficiency in estimating FCGR3 CNV by comparing Sequenom MassARRAY and paralogue ratio test-restriction enzyme digest variant ratio (PRT-REDVR). In addition, since many genetic association studies of FCGR3B CNV were carried out using real-time quantitative PCR, we have also included the evaluation of that method’s performance in estimating the multi-allelic CNV of FCGR3B. The qPCR assay exhibited a considerably broader distribution of signal intensity, potentially introducing error in estimation of copy number and higher false positive rates. Both Sequenom and PRT-REDVR showed lesser systematic bias, but Sequenom skewed towards copy number normal (CN = 2). The discrepancy between Sequenom and PRT-REDVR might be attributed either to batch effects noise in individual measurements. Our study suggests that PRT-REDVR is more robust and accurate in genotyping the CNV of FCGR3, but highlights the needs of multiple independent assays for extensive validation when performing a genetic association study with multi-allelic CNVs.

Comparison of Multiple Methods for Determination of FCGR3A/B Genomic Copy Numbers in HapMap Asian Populations with Two Public Databases

“…In three assay analysis, the concordant rates were much poorer, 62.22% for FCGR3B (TaqMan qPCR, PRT-REDVR and SYBR Green) and 60.00% for FCGR3 (TaqMan qPCR, PRT-REDVR and STR). This result was consistent with previous observations (Haridan et al, 2015). …”

Understanding the Genomic Structure of Copy-Number Variation of the Low-Affinity Fcγ Receptor Region Allows Confirmation of the Association ofFCGR3BDeletion with Rheumatoid Arthritis

“…In this context, genetic association studies at this locus that involve genotyping copy number, sequence variants, or both, can often be unreliable due to a combination of technical variation due to noisy assays measuring copy-number variation and biological variation because of the underlying complexity of the locus [Haridan et al, 2015;Hargreaves et al, 2015a]. Improved assays based on the paralog ratio test (PRT) [Armour et al, 2007] and multiplex ligation probe amplification (MLPA) [Schouten et al, 2002] have allowed some studies to suggest an association of the FCGR3B deletion with SLE [Morris et al, 2010;Niederer et al, 2010b] and RA [Robinson et al, 2012].…”

Discussion issues and new strategies of intravenous immunoglobulins treatment in neonatal practice

“…Это семейство бел-ков включает FcγRI (CD64), FcγRII (CD32) и FcγRIII (CD16) [21]. FcγRI-и FcγRIII-рецепторы относятся к активирующим рецепторам, а FcγRII -к ингибиру-ющим рецепторам [22].…”